Abstract

The development, health, and phenotype of monozygotic twins can be decisively affected by dietary behaviour.
Several channels of biochemical, microbiological, and physiological differentiation between twins are affected by the
particular traits of any given diet and are to be evaluated under a new perspective and point of view. The nutritional
factors have a more direct impact in the gut flora, obesity and its associated health problems. Diabetes and
cardiovascular diseases show a strong dependency on the dietary options often trumping the genetic aspects may
be affected leading to the metabolic syndrome and other diseases. Other systems such as the endocrine and the
immune systems also give further examples of the differentiation of health outcomes as a result of dietary patterns.
Moreover, cancer frequency, onset, and development are partially related to food constituents, especially in the case
of cancer diseases arising in the gastrointestinal tract. The epigenetic changes that occur during lifetime be partially
due to nutrition and may contribute to the pathogenesis of cancer. Indeed, some evidence ascribes phenotypic
discordance between monozygotic twins partially to epigenetic factors. However, the role of diet in the development,
ageing, and health status of monozygotic twins is still not fully understood and warrants further study. It is possible
that over the next decade a full characterization of human genomic, epigenomic, and transcriptomic data will be
within the reach of most researchers and shed much light onto the interplay of genetic determined processes and
nutrition effects.

Keywords

Monozygotic twins; Behaviour; Nutrition; Gut flora;
Health endpoints

Introduction

Unquestionably, diet has a large impact on the development, wellbeing,
and health of monozygotic twins, thereby decisively being one
of the most important factors modeling phenotypic variations between
them. This happens through diverse channels and according to very
distinct class of phenomena. Indeed, these encompass such different
aspects as the gut flora, the direct and indirect effect on cardiovascular,
metabolic, and endocrine physiology of the human body, the immune
system, challenging diseases as cancer, or very subtle changes at the
epigenetic level. All these sensitive areas together with the science
underpinning such realities are the subject of a thorough analysis in
this chapter. A final section deals with those subjects not totally
understood or still open to further research progress as well as with the
evolution perspectives for this particular field of scientific endeavor.

The role of the diet in the development of humans and their health
and the large impact of recent dietary changes are well known [1]. The
link between food and health has been evaluated and related with
scientific studies [1]. According to popular wisdom, “we are what we
eat”. Of course, this is exaggerated if it is to be interpreted as meaning
that diet determines all human phenotypical features and health. In
fact, genetics, biological factors such as the intrauterine environment,
upbringing, education, child and adolescent environment, pollution,
occupational hazards, social aspects and situations, and many other
factors exert a strong influence on the specific set of characteristics of
an human adult and its propensity to disease [2,3].

History tells us that a great importance was always attached to food
and human evolution was largely influenced by food resources and
strategies to use the nutrient-richest foods [4]. The agricultural
Neolithic revolution and the emergence of the so-called Western diet
have profoundly affected the nutritional balance of human diet and are
deemed responsible for several diseases [1]. Indeed, ‘seven deadly sins’
have been ascribed to the diet of sedentary human populations,
particularly, in the more developed countries: Glycemic load (blood
glucose raising potential), fatty acid composition (for instance, low ω3/
ω6 fatty acid ratios and an excess of saturated fat), macronutrient
composition (excessive fat and easily absorbed sugars), micronutrient
density (for instance, low zinc), acid-base balance (net acid generation
after metabolism), sodium-potassium ratio (too much sodium), and
fibre content (too low) [1]. Besides being perceived as an essential
condition for survival and health, early observers reported food
importance to mental or physical fitness [5].

In the particular case of twins, its study started in earnest in the XIX
century with the objective of estimating the relative powers of nature
and nurture [6] that is, the relative role of genetics and environment
(diet, education, etc.). Francis Galton’s ‘The history of twins’ was a
seminal work in this field [6]. This Victorian era researcher concluded
that heredity mattered more than environment. However, the
introduced and developed methodologies were more important than
this conclusion. As concepts evolved during historical time, these
methodologies were later further developed with the establishment of
the classical twin method of comparing the similarities of monozygotic
and dizygotic twins [7]. In this context, diet is a fundamental factor
that operates independently of genetics to a large extent, since it has
been shown that food preferences are primarily attributed to
environment and not genetic predispositions [8]. The first, systematic,
clinically-ascertained twin studies of the diet-genetics binomial are mainly found in the second half of the XX century, for instance,
regarding eating disorders [9] with first studies pointing to higher
concordance rates in monozygotic twins than in dizygotic twins [10].

The studies between monozygotic twins enable to exclude the
genetic factor as a variable and, as such, are very useful whenever
environmental factors for instance, diet-need to be examined. This may
be very important when critical issues and the connections between
diet and obesity [11], diabetes [12], cardiovascular [13-15], metabolic
[16], endocrine [17] immune system [18] and cancer [19] diseases are
to be highlighted. Accordingly, the gold standard for distinguishing
genetic from environmental traits has been the comparison between
twins [20]. The following sections will cover these several connections
and systems, thereby highlighting the complex interplay between
genome, epigenetic factors, and nutrition and underlining how much a
given diet may differentiate the nature and fate of monozygotic twins.

Gut Flora: A Factor of Variability between Twins

It is well known that human gut flora as well as other human
microbiomes is quite rich and diverse [21]. The area most colonized by
microbes is the distal gut [22]. The typical gut flora alone consists of
hundreds of bacterial species, collectively encoding an enormous gene
set that is 150 times larger than the human set of genes [23]. The gut
microbiome is fundamental for many essential processes, including
vitamin and amino acid biosynthesis, dietary energy harvest, and
immune development [24]. This importance and the observed large natural variability leads to an obvious question: does gut flora differ
between monozygotic twins?

It must be remarked that human microbiome projects are being
initiated throughout the world, with the goal of correlating human
physiological phenotypes with the structures and the functions of their
indigenous microbial communities [25]. These authors have
endeavoured to sequence in a more thorough way the DNA of the
organisms composing the gut microbiome. This was also done with the
purpose of understanding how much of the observed organismal
diversity is due to methodological insufficiencies. Indeed, it was
confirmed a high level of species diversity after reduction of
methodological noise [25]. It was shown that the 54 studied twin pairs
had very different species assemblages (Table 1). There were important
differences at the genetic microbiome level and at other biochemical
levels. Namely, differences within twins were found in the set of
carbohydrate active enzymes (for instance, cellulases) and in
transcriptional activities [25]. It was observed that the microbial
community genes encoding for the carbohydrate-active enzymes were
highly enriched in the Faecalibacterium bins found in the microbiome
of one of the twins, but not in the other. Even genes widely distributed
could lead to variability as a result of abundance variation. Therefore,
results highlighted another level of genetic variation between humans,
imparting variability to otherwise genetically identical twins [25].
Though these twins were at least 5 km apart [26], they were both obese
and had a very similar life history: they had been vaginally delivered
and had no history of intestinal disease.

Health Aspect

Main Findings

Reference

Gut flora

Twin pairs had very different species assemblages, different carbohydrate active enzymes, and different transcriptional activities

Table 1: Main findings in the scientific literature concerning dietary effects on the health and well-being of monozygotic twins.

Of course, physical separation meaning living in different
households and environments and experiencing slight differences in
diets and other aspects seems to be important in generating human
microbiome diversity in monozygotic twins. For instance, an
interesting study [27] showed that commercially available fermented
milk products (FMP) containing a consortium of bacterial strains,
such as Bifidobacterium animalis , Lactobacillus delbrueckii ,
Lactococcus lactis and Streptococcus thermophilus , were able to have
an impact in the human fecal metatranscriptome though confined to
the period of FMP consumption. However, no significant effect was
detected on the bacterial species composition or in the proportional
representation of genes encoding known enzymes [27]. Hence, effects
were circumscribed to changes at the expression level of microbiomeencoded
enzymes involved in several metabolic routes, namely
concerning carbohydrate metabolism. A different caloric content in
the diet over many years may not only lead to different outcomes
concerning obesity, but also affect the composition and characteristics
of the human gut microbiome [22] Such effects were identified in a
dataset composed of twin-mother trios [22].

28]. This was shown for ileal Crohn’s disease.
These diseases are only to a limited extent affected by human genetics
[29] genetic susceptibility, being common twin pairs discordant for
disease [28]. In fact, concordance rates for monozygotic twins range
from 6 to 17% for ulcerative colitis [30-33] and from 37% to 58% for
Crohn’s disease [31,33]. Concerning this IBD subject, significant
differences in the gut microbiomes of identical twins according to
Crohn’s disease status have been found [34,35]. Hence, differences in
diet that may cause specific bacterial assemblages in the GI tract are
conducive (together with other factors) to higher probability of
developing IBD and thus become a source of variation in the quality of
living of monozygotic twins. Nevertheless, studies are not fully
conclusive and the dietary aspects leading to IBD are not clearly
identified [36]. There is some evidence associating a higher intake of
ω6 fatty acids [37] as well as frequent fast-food intake [38] with
enhanced IBD risk.

Future research in this novel scientific field is needed. Particularly,
the characterization of the enzymatic activity of these systems, the
breadth of their distribution of organisms, the host and environmental
parameters (namely diet) determining their abundance in the human
gut microbiome, and their effects on host nutrient/energy budget are
all aspects whose study is warranted.

Obesity, Diabetes and Cardiovascular Disease:
Nutrition and Twins

Overweight and obesity are an important worldwide concern in
terms of clinical and public health since they are associated to an
increased risk of other diseases, such as type-2 diabetes and cardiovascular diseases (CVD), which are major causes of mortality
[39]. In 2005, the estimated world’s overweight adult population were
937 million and the obese reached the 396 million. Yet, it is expected
that in 2030 the overweight and obese adult’s figures surpass a total of 2
and 1 billion of individuals, respectively [39]. In what concerns
children, the prevalence has augmented significantly in developed and
developing countries [40]. It has been estimated that overweight and
obesity caused more than 3 million deaths worldwide in 2010, with
huge public health losses as measured in disability-adjusted life-years
(DALYs) [40].

It is well known that the onset of obesity is triggered by an
imbalance between energy intake and expenditure [41] and that the
quantity of calories ingested has a direct impact on human weight.
Indeed, there are several nutritional recommendations to prevent
weight gain and other diseases, like CVD and type 2 diabetes. In fact, it
has been recommended the consumption of whole grains, vegetables,
fish, fruits, and nuts, whereas the consumption of refined grains and
sugary drinks is deemed pernicious.

Twin studies have shown that there are genetic influences on obesity
[42]. Twin studies estimate heritability of body mass index (BMI) to be
40-70% in children and adults [43,44] (Table 1) and other
anthropometric measures of obesity and fat distribution, such as
skinfold thickness, waist circumference, and waist to hip ratio display a
similar impact of heritability [43-46]. Twins studies have also demonstrated a considerable influence of the genes on eating patterns
of adults [47].

On the other hand, nutrition seems to be decisive for weight
variation and obesity outcomes [48]. Of course, shared environmental
influences during the infancy of twins contribute to reinforce the
genetic driver to similarity between twins [49]. However,
environmental differences after leaving their parents’ home may
generate important differences, namely concerning obesity [50]. This
study on monozygotic twins highlighted the great importance of diet
for obesity development. In fact, the obese twins reported preference
for fatty foods three times more frequently than the lean co-twin [50].
Furthermore, when recalling taste preference for fat at the time the
twins left their parental homes, both the obese and lean co-twins
consistently recalled that the obese twin had greater preference for
fatty foods than the lean twin. Otherwise, psychological traits of the
lean and obese co-twins did not diverge [50]. Hence, the conclusion is
that preference for fatty foods and consequentially a diet richer in fat
leads to obesity regardless of the genetic background. This preference
may be due to several different factors, being possible that education
exerts some effect on BMI [51].

Food preferences, dietary patterns, and obesity are influential in the
development of type 2 diabetes. As with obesity, genetically based
processes and environmental influences are both important. It has
been claimed that heritability is high for slowness in eating (over 80%
share) and satiety responsiveness (over 65%) and not so high for food
responsiveness [less than 65%) [52]. A prospective study on
monozygotic twins reported that the observed rate of concordance for
type 2 diabetes can reach, at least, 76%, thereby pointing to a very
strong genetic component [53]. However, the fact that not all
monozygotic twins are concordant for this disease suggests that
environmental factors may be relevant. A review [12] has established
that an increased risk for developing type 2 diabetes is associated with
overweight and obesity, abdominal obesity, physical inactivity, and
maternal diabetes. These authors also found probable that a high
intake of saturated fat contributes to an increased risk. Moreover, they
claimed that from existing evidence it is also possible that n-3
polyunsaturated fatty acids (n-3 PUFA) and low glycemic index foods
may reduce disease risk, while total fat intake and trans fatty acids may
enhance risk [12]. It should also be remarked that the monochorionic
intrauterine nutrition of most monozygotic twins has been shown to
favor growth retardation [54] and that low birth weight is associated
with increased risk of type 2 diabetes later in life [55,56].

Obesity is also a risk factor for CVD [57]. Data showed that weight
gain after the young adult years conveyed an increased risk of CVD in
both sexes that could not be ascribed either to the initial weight or the
levels of the risk factors that may be due to weight gain. Besides
obesity, there are other risk factors for CVD that can be directly-for
instance, excessive salt [58] or indirectly diabetes [59] linked to diet.
Nevertheless, genetic factors also play a role [15]. For instance, a study
on the genetic and environmental effects on susceptibility to heart
diseases for males and females with data from monozygotic and
dizygotic twins confirmed the existence of an important role for
genetics (Table 1) [60]. Indeed, it was shown that individual
susceptibility to mortality due to heart diseases and coronary heart
diseases had a strong genetic influence in both males and females.

However, studies on monozygotic twins have also shown that
genetic factors do not explain all, being diet a decisive factor [61]. The
influence of nutrition starts in the womb [62]. It has been proposed
that fetal malnutrition in middle to late gestation may lead to permanent changes in metabolism and physiology that raise the risk of
CVD in adulthood [63]. But, intrauterine nutrition is as shared as
genes by the monozygotic twins and does not act as a differentiating
variable.

Among nutritional parameters, mounting evidence suggests that too
much sodium in a diet is conducive to a higher risk of stroke and CVD
[64,65]. This was corroborated by a recent study [61] involving 286
male middle-aged twins. It was shown that habitual dietary sodium is
inversely associated with coronary flow reserve independent of other
factors, thus meaning an adverse effect of sodium on the
cardiovascular system [61]. Regarding this issue, a recent study
involving twins pointed out that both genetic predisposition and
shared environment contribute to sodium intake [66]. However, salt
consumption habits must be an informed choice of each individual.

It has also been reported that n-3 PUFA typically found in fish and
other seafood have a positive effect on the lipoprotein profile [67]. This
study on monozygotic twin pairs showed that n-3 PUFA increased the
high-density lipoprotein 2b, which is deemed to be protective against
CVD. Conversely, another twin study established a connection
between a sweet-laden and fatty diet poor in n-3 PUFA and a
pernicious variation of the triglyceride levels as well as of the particle
size of very low-density lipoprotein [68]. Such effects were brought
about by a ‘junk food’ diet encompassing French fries, hamburger,
pizza, salty snacks, and sweets.

Therefore, though genetic, intrauterine, and infancy environment
factors have an effect on the cardiovascular system, CVD is not
predetermined by genes, being possible by choosing a healthy fat and
low sodium diet to reduce disease risk. Furthermore, the inclusion of nutraceuticals in the diet may counteract dyslipidemia and reduce
cardiovascular risk factor for coronary heart disease [69]. In this way,
two identical twins may experience very different health outcomes if
they choose different diets.

Nutrition and its Impact on Metabolic and Endocrine
Diseases in Twins

The relationship between nutritional aspects and the development
of metabolic and endocrine diseases is another important area of
research also involving monozygotic twin studies.

Some of these diseases are related to obesity, diabetes, and CVD,
which were addressed in previous section. Namely, the metabolic
syndrome, a problem affecting energy utilization and storage, is
considered to increase the risk of developing type 2 diabetes mellitus
and CVD [70]. There is convincing evidence that diet plays an
important role in the development and progression of the metabolic
syndrome. Obesity is a key aetiological factor in the development of
this health problem [70]. Monozygotic twin studies help
understanding the biological impact of gene–nutrient interactions and
provide a key insight into the pathogenesis and progression of dietrelated
polygenic disorders, including the metabolic syndrome [16,70]
The difference in concordance rates between monozygotic and
dizygotic twins shows that this disease is at the crossroads of diet and
genetics. In contrast, the heritability estimates for hyperinsulinaemia, hypertension, and hypertriacylglycerolaemia are low, thus pointing to a
larger role of environmental influence on these components of the
metabolic syndrome [16]. Several twin studies have emphasized the
importance of environmental factors in different aspects related to
metabolic syndrome [71,72] thereby circumscribing the impact of
genetic factors to approximately 50% (Table 1) [72].

With regard to endocrine system diseases, there are quite diverse,
ranging from diabetes mellitus (previously addressed) to goiter (largely
related to iodine deficiency) and other thyroid diseases [some also
autoimmune such as Graves’ disease and Hashimoto’s thyroiditis),
Cushing’s disease, or premature ovarian failure. The relative
importance of diet, other environmental factors, and genetic causes in
the pathogenesis of this diverse array of diseases varies widely.
Moreover, the interaction between environmental and genetic factors
may be quite complex as in the case of Graves’ disease, which is a
specific form of hyperthyroidism [73].

In the case of goiter, a strong genetic dependence is indicated by
twin studies [74]. There is a higher concordance rate (over 50%) for
goiter in monozygotic than in dizygotic twins [75]. Nevertheless,
important influence is exerted by the diet, either by the iodine content
in food or the presence of goitrogenic constituents in diet, such as
flavonoids in millet and soybean, cyanogenic glucosides in cassava, or
glucosinolates in vegetables belonging to the genus Brassica [76].

Sensitivity of the Twins Immune System to Dietary
Variation and Autoimmune Disease

The immune system of humans and in particular, of monozygotic
twins also responds to dietary patterns and their changes. As it is well
known, this system has evolved to protect us from disease caused by
microorganisms. This requires differentiation between materials that
belong to the human organism (self) and those that do not belong
(non-self). In some cases, there is a defective reaction of the immune
system and so-called autoimmune disease develops as a result of
reactivity to self. Complex interactions between genome and the
environment determine which individuals will be affected by any given
autoimmune disease. Important diseases in this class include celiac
disease, lupus erythematosus, multiple sclerosis, psoriasis, rheumatoid
arthritis, Sjögren’s syndrome, vasculitis, Hashimoto’s thyroiditis, and
many others.

The influence of environmental factors on the genesis of these
diseases has been shown by studies on identical twins [18]. For
instance, these twins have disease concordance rates of only 50%
(Crohn’s disease) or less (30%, for multiple sclerosis) (Table 1). In
multiple sclerosis (MS), environmental factors such as vitamin D
intake [18] and sunlight exposure [77] seem to be important as shown
by comparison between monozygotic twins. A Danish study on
monozygotic twins has also shown that besides genetic factors
environment is of etiological importance for the genesis of
autoimmune thyroid diseases [78]. Among environmental variables,
diet can strongly influence the human body and its susceptibility to
disease [79]. The mechanisms for this influence may be direct or
indirect, even involving epigenetic changes (see below).

Concerning MS, there is a gradient of growing risk with higher
latitude, which is coincident with sunlight exposure reduction and
more frequent vitamin D deficiency [80] Vitamin D supplementation
[81] and high concentrations of serum 25-hydroxyvitamin D in
adulthood [82] were linked to lower MS risk. The importance of the
latter substance was corroborated by a twin study approach [83].
However, in this case, gene factors may influence the trait.
Nevertheless, the importance of dietary factors in the genesis of MS
seems to be significant-consumption of fish three or more times a
week was protective against MS for individuals with low sunlight
exposure [84] and further studies on twins, especially using a casecontrol
approach have been advised [85].

With regard to rheumatoid arthritis, there is a clear genetic
component [86], but the concordance rate in monozygotic twins does
not exceed 30%, leaving 70% to be explained by other factors [87,88].
The severity of the disease may also differ among concordant twins
[89]. Dietary factors have been suggested to play a role [90]. Namely,
higher consumption of caffeine and red meat has been associated with
an increased risk of rheumatoid arthritis. On additionally, a higher
level of intake of cooked, but not raw, vegetables has been claimed to
protect against the onset of disease [91]. Moreover, a randomized
placebo-controlled trial conducted on twenty pairs of identical twins
over a period of six months showed that dietary supplementation with
calcium and vitamin D greatly improved bone mineral density, a
parameter that is associated to the severity of rheumatoid arthritis and
that worsens life quality of patients [92].

Type 1 diabetes is also an immune-mediated disease, since β cell
destruction is due to autoimmune reactivity [93]. Scientific evidence
such as low disease concordance (less than 40%) in identical twins
points to a critical role of environment in the disease etiology [94]. Nutritional factors are thought to be of high importance [95] Gut
incretin hormones are secreted in different amounts as a response to
glucose in twins with the disease, but not in their healthy co-twins [96]
Increased weight gain in infancy has been associated to greater risk of
type 1 diabetes. Moreover, cow milk is considered diabetogenic [97].
On the other hand, some vitamins and minerals have been proposed to
protect against type 1 diabetes [95]. Recent results [98] have shown
that Nε-carboxymethyl-L-lysine (CML), a glycotoxin possibly related
to heat-treated dietary factors [99], is a diabetes risk factor. In twins,
familial environment explained 75% of CML variance, thus pointing to
the importance of diet in the development of disease.

For the Hashimoto’s thyroiditis, concordances rates varying widely
between 35% and 70% in identical twins have been reported [100],
thus suggesting a role for nutritional factors. However, the
identification of possible dietary parameters that enhance the risk of
this and other endocrine system diseases is still in its infancy.

In general, immune system studies using twin pairs have
highlighted the relative importance of genetic and environmental
factors. For instance, regarding food allergies not mediated by
immunoglobulin E (IgE), high concordance rates of approximately
75% among identical twins have been reported in different studies
[101] Despite this high value, it is considered that environmental
factors are also decisive in these and other (IgE-mediated) food
allergies. Indeed, various twin studies stress the importance of genome,
but show that there is significant variation due to environmental
exposure [102].

Diet as a Fundamental Cause Affecting Cancer in Twins

The effect of diet on the generation of cancer is considered
significant along with other factors [103,104]. Indeed, important
organizations such as the American Cancer Society have encouraged
changes in diet, namely lower saturated fat and higher fruit and
vegetable consumption [103]. There are several important dietary
substances, which may be influential in the incidence of cancer, such
as, resveratrol (in red grapes and berries), genistein (soybean), allicin
(garlic), lycopene (tomato), β-carotene (carrots), and dietary fibre
[105]. However, epidemiological available data are not consistent for
many foods, remaining associations with cancer risk unclear [106].
Owing to the impact on some critical aspects (obesity, ω3/ω6 fatty
acids ratio, meat consumption, polyphenol and sulphur compounds, vitamins, minerals, isoflavones, fibre and others), dietary options may
lead to an enhanced or reduced cancer risk [106].

Therefore, dietary factors are expected to lead to different cancer
risk in identical twins if these chose distinct diets over a long time
period. On the other hand, as was previously mentioned, studies on
monozygotic twins eliminate the genetic factor as a variable and allow
the assessment of the importance of environmental factors. However,
there are some shared environmental background aspects in identical
twins such as smoking or diet in childhood family [107]. Even though
part of the diet effect may act as a non-differentiating factor, whenever
genetically identical twins move to distinct surroundings and have
different diets, cancer risk factors may change [107]. Indeed, a study on
cohorts of twins from Sweden, Denmark, and Finland [107] has
concluded that environment has the main role in causing sporadic
cancer (Table 1) [especially for colorectal and breast cancer), being the
contribution of inherited genetic factors considered minor to most
types of neoplasms. Nevertheless, there are some cancer diseases
(prostate and colorectal), which show statistically significant effects of
heritability [107].

The cancer diseases related to the GI tract are the most obvious
connection between diet and carcinogenesis. Important factors in the
genesis of cancer like Helicobacter pylori infection for gastric
carcinoma [108] are independent of the genetic makeup [109]. These
authors found that among monozygotic twins reared apart and
discordant for H. pylori status, infected twins consumed more ascorbic
acid than their unaffected co-twins. GI carcinogenesis can also be
induced by obesity [110]. The mechanisms whereby obesity leads to
several types of GI cancer diseases (particularly, esophageal, gastric,
pancreatic, and colorectal) involve changes in insulin levels, adipokines
secretion, inflammatory cytokines contents and other important
physiological parameters [110]. Diverticular disease is another
potential GI carcinogenic factor that is partially caused by low dietary
fibre [111]. Though results of a twin comparison by these authors show
that 53% of susceptibility to diverticular disease is ascribable to genetic
factors, a significant impact of food choices is acknowledged.
Furthermore, the risk for esophageal adenocarcinoma is increased by
gastroesophageal reflux disease, which in turn was shown to be a
condition sensitive to BMI and, as such, dietary aspects, in an
important study involving monozygotic twin pairs [112].

Gut microbiota may also influence the incidence of cancer, namely
colorectal cancer [113]. As mentioned above (section 2), diet may
model the gut microbiota [114] and as such, produce an effect on
cancer risk. Hence, dietary options may be a cause for a differential
cancer risk and mortality in monozygotic twins. There are other
indirect connections between diet and cancer. Namely, DNA is
methylated over time and monozygotic twins’ studies show that the
methylation status diverges with age, thereby demonstrating that this
phenomenon is susceptible to environmental factors [115]. These DNA
changes may have an important role in cancer [116] and are affected by
diet and nutrient intake [117,118]. It should be emphasized that such
DNA alterations may be relevant for cancer diseases other than GI
cancer. This issue is further developed below in the diet and epigenetics
section (section 7).

There are also cancer types where the case for a dietary effect is
weak, such as testicular cancer [119]. These authors found factors other
than diet causing a difference in cancer incidence in twins. For other
cancer diseases, it is difficult to separate the genetic and environmental
factors through monozygotic twins’ comparison because several
prenatal aspects are intertwined. For infant leukemia, the connection between maternal diet and disease remains a hypothesis [120]. It is
assumed as possible that dietary exposure to substances that inhibit
topoisomerases could lead to disease. However, it has been only shown
a prenatal origin for some childhood leukemias [121,122].

All these studies and the associated progress made on the
understanding of the cancer process point to the importance of
primary prevention including a healthy diet as the most effective way
to reduce risk and mortality of some types of cancer [123].

Food as a Source of Epigenetic Change

Epigenetic changes are heritable phenotypic traits that are not
caused by changes in the DNA sequence and are of great importance
for individual development and disease. The epigenetic changes are
preserved when cells divide. In this context, food constituents may act
upon several biochemical phenomena at the cell nucleus, thereby
affecting gene transcription and modulating gene expression [124].
Nutrients needed for nucleic acid synthesis and for the associated
regulating enzymes are especially interesting: essential amino acids,
zinc, folate, and vitamins B6 and B12 [125]. In fact, it has been claimed
that epigenetic change during individual development to be stochastic
and/or determined by environmental factors. Namely, these epigenetic
phenomena encompass histone modification or DNA methylation
levels, which change over time [115]. Monozygotic twins’ studies may
be very helpful for shedding light into this subject, since monozygotic
twins are genetically identical [79]. These twins have the same
genotype because they are derived from the same zygote. Nevertheless,
monozygotic twin siblings may display many phenotypic differences,
such as their susceptibility to disease and several anthropomorphic
traits. Indeed, there is some evidence that phenotypic discordance
between monozygotic twins is partially due to epigenetic factors (Table
1) [124].

For elder twin pairs, differences in gene expression were reported to
be four times greater than those in younger twin pairs [115]. There
seems to be an age-dependent accumulation of epigenetic differences,
thereby suggesting the existence of the so-called epigenetic drift.
Particularly, older monozygotic twins displayed remarkable differences
in their overall content and genomic distribution of 5-methylcytosine
DNA and histone acetylation [115]. It must be remarked that
epigenetic changes regulate several genomic effects, including the
expression of genes fundamental for normal growth, development, and
differentiation, without affecting the DNA sequence, and, differently
from the fixed DNA sequence, show significant plasticity [124].

Lower levels of methylation (hypomethylation) were associated with
the overexpression of repeated DNA sequences that must be repressed
in healthy cells [124]. Hypermethylation typically involves cytosinephosphate-
guanine islands in the promoter region and it is linked to
gene inactivation. Indeed, hypermethylation is one of the most
important epigenetic changes repressing transcription via the promoter
regions of tumour suppressor genes [126]. Global hypomethylation is
also associated to the development of cancer by different pathways
[126]. Diet can both prevent and induce colon carcinogenesis through
epigenetic changes, which regulate the homeostasis of the intestinal
mucosa [127]. Therefore, the study of epigenetics, such as the DNA
methylation profiles in monozygotic twins, is quite important [128].
Recent methodological progress (through methylated DNA
immunoprecipitation followed by deep sequencing) has enabled to
make significant advances and carry out large cohort twin studies, which may help to explain the mechanisms linking diet and epigenetic
changes [129].

Some experimental works [130,131] showed that DNA methylation
was influential on the regulation of the glucose transporter 4 and leptin
genes during adipocyte differentiation. So, there may be a connection
between epigenetic phenomena and obesity. In fact, a study on obesitydiscordant
monozygotic twins has shown several differences in the
transcription profiles in adipose tissue between twins [132]. The results
showed the effects of acquired human obesity, which may be related to
epigenetic modulation of the genome.

It has also been reported that epigenetic mechanisms are involved in
the etiology of age-related macular degeneration (AMD) and that such
mechanisms link specific dietary factors and AMD in monozygotic
twins [133]. Indeed, the twin with the less advanced AMD displayed
frequently a higher dietary intake of vitamin D, betaine, or methionine
[133]. Some study results [134] seem to point to randomness as a cause
for epigenetic alterations correlating with discordance for disease
among monozygotic twin pairs. However, it is quite possible that
dietary differences, even if small, may be a driving force [135]. Hence,
the complexity of this scientific field and the intricacies deriving from
multiple environmental factors and affecting monozygotic twins entail
that diet-epigenetics connections require further study.

Open Issues and Future Perspectives

The studies presented in the previous sections provide some insight
into the importance of nutrition for the development, ageing, wellbeing,
and health status of monozygotic twins (Figure 1). A summary
of the more direct and clearly circumscribed links between nutrition
and twin health can be seen in Table 2. Nevertheless, there are several
issues requiring further study. Namely, a better understanding of the
importance of genetics, epigenetics, relation with environmental
aspects, and other factors as well as of the synergies between these
aspects can be attained from novel monozygotic twin studies. The
observed phenotypic differences between genetically identical twins
highlight the relationship between genetic determinants and
environmental factors [79]. The epigenetic changes bring another level
of complexity to the study of interactions between the environment,
and in particular diet, and phenotypic traits with special emphasis on
disease susceptibility. Twin studies may offer the opportunity to study
epigenetic variation across the genome. On the one hand, these studies
can improve the understanding of the factors regulating epigenetic
variability by assessing the heritability of epigenetic variants [136]. On
the other hand, the use of twins in epigenetic research may help to
unravel the intricacies associated to human development and disease
and make their connections clearer, thereby exposing the role of
nutrition.

Figure 1: The multiple channels through which nutrition affects the
health status.

A recent study [14] on monozygotic twins found that a
Mediterranean diet led to a lower level of oxidative stress, as measured
by the glutathione redox pair, thus offering a possible mechanism
linking diet and CVD. This is a good example of future randomized
controlled trial studies on diet and health outcomes using identical
twins as a population where the genetic variability is absent.
Furthermore, future scientific work should focus more on age-related
traits and diseases and try to link phenotypic differences between
monozygotic twins to environmental differences and epigenetic factors
[137].

The ageing of populations and the recent great shifts in dietary
patterns of large swaths of the world population have decisively
contributed for the emergence of new important diseases that were
rare one hundred years ago [70,138,139]. Alzheimer disease and other
ageing related health conditions belong to this group of emerging
diseases and the conduction of trials using identical twin pairs may
provide new insights [138]. Other disease group is more directly
associated with nutritional factors and encompasses CVD, diabetes,
and cancer [139]. The clearer identification of the critical nutritional
factors also makes advisable the conduction of more studies on
monozygotic twins.

It is highly plausible that over the next decade a full characterization
of human genomic, epigenomic, and transcriptomic data will be within
the reach of most researchers [136]. This advance may shed much light
onto the interplay of genetic determined processes and nutrition
effects. It will become much clearer to what extent genes determine
human traits and disease and the scope of action of dietary parameters,
thereby highlighting how much a given diet may differentiate the
nature and fate of monozygotic twins.

Acknowledgments

This work was supported by the following Post-Doctoral Grants:
Ref: SFRH/BPD/102689/2014 (“Fundação para a Ciência e a
Tecnologia”, FCT) for the author Carlos Cardoso; Ref: SFRH/BPD/
64951/2009 (FCT) and DIVERSIAQUA (MAR2020) for the author
Cláudia Afonso” (FCT).

Conflict of interest

No conflict of interest. This research received no specific grant from
any funding agency, commercial or not-for-profit sectors.